Selective Glycopeptide Signal Enhancement Using the Athena Ion Processor in the Collision Cell on a timsUltra AIP system

Significance of the Topic

Glycopeptide characterization by mass spectrometry is essential for understanding protein glycosylation in biology, biopharma, and disease biomarker discovery. However, glycopeptides ionize inefficiently, produce low precursor signals and labile glycan fragments, and often yield poor MS/MS spectra that complicate confident glycan structure assignment. Hardware-level improvements that selectively boost transmission of glycopeptide ions across specific mass-to-charge (m/z) and ion mobility (IM) windows can increase sensitivity without sacrificing throughput or consuming more sample. This study evaluates an RF-based ion-manipulation module, the Athena Ion Processor (AIP), integrated into the timsTOF Ultra collision cell, to address these analytical challenges.Objectives and Study Overview

• Systematically optimize AIP operating parameters (RF amplitude, slope of the RF/mobility ramp, base storage time) to selectively enhance transmission of glycopeptide precursors and diagnostically important fragment ions.
• Assess effects on precursor intensity, oxonium and Y ion abundances, and overall MS/MS spectral quality using a glycoprotein standard and complex biological samples.
• Demonstrate practical gains in glycopeptide identification from HILIC-enriched HeLa digests using the optimized AIP workflow compared with a standard Ultra2 acquisition.

Methodology and Instrumentation

• Samples: Alpha-1-acid glycoprotein digest (10 ng/µL) used for parameter optimization by direct infusion; HILIC-enriched N-glycopeptides from HeLa cell digests (100 ng injection, 30-min nanoLC gradient) for application testing.
• Chromatography and MS: nanoElute nanoLC coupled to a timsTOF Ultra equipped with the Athena Ion Processor (AIP) implemented inside the collision cell. Experiments included targeted MRM-style MS/MS to evaluate spectral quality and targeted m/z range scans filtered for z>1.
• AIP parameter space explored: RF amplitude (kVpp), RF frequency and linear AC ramps, slope shapes (steep, broad, exponential variants), and base storage time including mobility-guided ramping. TOF pulse timings and storage windows were adjusted to favor transmission of specific m/z and IM populations.
• Data interrogation: monitored precursor ion intensities, diagnostic oxonium ions, b/y peptide fragments, larger-mass Y ions, and computed glycoPSMs, unique glycopeptides and glycoprecursors to quantify identification gains.

Main Results and Discussion

• Parameter optimization on the alpha-1-acid glycoprotein standard established that combinations of increased RF amplitude, appropriately shaped RF/mobility slopes and longer base storage times could concentrate transmission in targeted m/z/IM regions. Larger RF and steep slopes focused transmission toward higher m/z species, while broader slopes allowed simultaneous passage of low-mass oxonium ions, medium b/y fragments and high-mass Y ions.
• Mobility-guided base storage time proved effective: longer storage preferentially retained larger ions (higher m/z), and using an IM–mass heat map guided selection of base storage timing improved selectivity for glycopeptide precursors.
• Representative MS/MS spectra comparing optimized AIP conditions versus no-AIP showed marked increases in Y-ion intensities and improved overall fragment ion richness under AIP, enhancing confidence in glycan structural assignment.
• In a 30-minute nanoLC run of HILIC-enriched HeLa samples, the optimized AIP workflow produced substantial identification gains versus the Ultra2 reference: glycoPSMs increased by 42.3%, unique glycopeptides by 38.6%, and unique glycoprecursors by 42.4%. These improvements coincided with higher precursor intensities and better-quality MS/MS spectra.

Benefits and Practical Applications

• The AIP provides selective, hardware-level enhancement of glycopeptide signal without requiring larger sample loads or longer gradients, preserving throughput and sample economy.
• Improved Y-ion transmission directly benefits glycan composition and topology determination, reducing manual interpretation and increasing automated identification confidence in glycoproteomics workflows.
• Mobility-guided ramping allows targeted enhancement across co-eluting species having distinct IM distributions, enabling more effective prioritization of glycopeptide precursors in discovery and targeted workflows.
• Applicable contexts include basic glycoproteomics research, biotherapeutic glycoform characterization, biomarker studies, and method development for clinical proteomics where sensitivity and spectral completeness are critical.

Future Trends and Potential Applications

• Integration of AIP-style selective transmission with advanced acquisition schemes (e.g., data-independent acquisition adapted for IM-resolved windows) could further increase depth and reproducibility of glycoproteome coverage.
• Adaptive or real-time optimization of AIP parameters driven by on-the-fly IM and m/z profiling could maximize signal for diverse glycoforms across complex samples.
• Combining AIP enhancement with complementary fragmentation (e.g., ETD/EThcD) may yield richer peptide backbone and glycan-specific fragments, improving site-specific glycosylation mapping.
• Wider adoption may stimulate standardized parameter sets or vendor-supplied AIP profiles for common glycopeptide classes and LC setups, easing transfer between laboratories.

Conclusion

Systematic tuning of the Athena Ion Processor within the timsTOF Ultra collision cell selectively enhances glycopeptide precursor and fragment ion transmission across defined m/z and ion mobility windows. Optimized RF amplitude, slope and base storage time increase precursor abundances and notably improve Y-ion intensity and overall MS/MS spectral quality. These hardware-level gains translate into substantial increases in glycopeptide identifications from HILIC-enriched HeLa samples while preserving fast nanoLC throughput and low sample consumption, offering a practical route to more sensitive and confident glycoproteomics.

Instrumentation Used

• timsTOF Ultra mass spectrometer with integrated Athena Ion Processor (AIP) in the collision cell
• nanoElute nano-flow liquid chromatography system
• HILIC enrichment for N-glycopeptide sample preparation
• Standard glycoprotein alpha-1-acid glycoprotein for direct infusion optimization

References

• Authors and affiliations: Hongxia Bai, Diego Assis, Kristina Marx, Benjamin Jones, Michael Krawitzky, Matthew Willetts; Bruker (Billerica, MA; Bremen, Germany; San Jose, CA).
• Study reported by Bruker; authors are employees and may hold financial interests in the company. For research use only; not for diagnostic procedures.